Soluble tumor necrosis factor-alpha-induced hyperexcitability contributes to retinal ganglion cell apoptosis by enhancing Nav1.6 in experimental glaucoma

Abstract

Background Neuroinflammation plays an important role in the pathogenesis of glaucoma. Tumor necrosis factor-alpha (TNF-α) is a major pro-inflammatory cytokine released from activated retinal glial cells in glaucoma. Here, we investigated how TNF-α induces retinal ganglion cell (RGC) hyperexcitability and injury. Methods Whole-cell patch-clamp techniques were performed to explore changes in spontaneous firing and evoked action potentials, and Na+ currents in RGCs. Both intravitreal injection of TNF-α and chronic ocular hypertension (COH) models were used. Western blotting, immunofluorescence, quantitative real-time polymerase chain reaction (q-PCR), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) techniques were employed to investigate the molecular mechanisms of TNF-α effects on RGCs. Results Intravitreal injection of soluble TNF-α significantly increased the spontaneous firing frequencies of RGCs in retinal slices. When the synaptic transmissions were blocked, more than 90% of RGCs still showed spontaneous firing; both the percentage of cells and firing frequency were higher than the controls. Furthermore, the frequency of evoked action potentials was also higher than the controls. Co-injection of the TNF-α receptor 1 (TNFR1) inhibitor R7050 eliminated the TNF-α-induced effects, suggesting that TNF-α may directly act on RGCs to induce cell hyperexcitability through activating TNFR1. In RGCs acutely isolated from TNF-α-injected retinas, Na+ current densities were upregulated. Perfusing TNF-α in RGCs of normal rats mimicked this effect, and the activation curve of Na+ currents shifted toward hyperpolarization direction, which was mediated through p38 MAPK and STAT3 signaling pathways. Further analysis revealed that TNF-α selectively upregulated Nav1.6 subtype of Na+ currents in RGCs. Similar to observations in retinas of rats with COH, intravitreal injection of TNF-α upregulated the expression of Nav1.6 proteins in both total cell and membrane components, which was reversed by the NF-κB inhibitor BAY 11-7082. Inhibition of TNFR1 blocked TNF-α-induced RGC apoptosis. Conclusions TNF-α/TNFR1 signaling induces RGC hyperexcitability by selectively upregulating Nav1.6 Na+ channels, thus contributing to RGC apoptosis in glaucoma.

EphA4/ephrinA3 reverse signaling mediated downregulation of glutamate transporter GLAST in Müller cells in an experimental glaucoma model

“…Activated Müller cells could induce microglia activation and the interaction of Müller cells and microglia aggravated retinal inflammatory response, which contributed to RGC injury in COH retina (Hu et al, 2021). Based on the present results and our previous study (Cheng et al, 2021; Hu et al, 2021; Xu et al, 2020), we speculate that at the early stage of IOP elevation, inhibition of the reverse signaling could rescue GLAST downregulation and attenuate Müller cell gliosis, thus relieving the activation of retinal glial cells and reducing retinal inflammatory response, which indirectly contribute to reduction of RGC apoptosis. On the other hand, inhibition of the forward signaling in RGCs may directly attenuate RGC apoptosis (Xu et al, 2020).…”

Sensitization of colonic nociceptors by TNFα is dependent on TNFR1 expression and p38 MAPK activity

“…2; P = 0.0001, one‐way ANOVA with Holm–Šídák's multiple comparisons test; n = 5−6, N = 5−6). Pre‐treatment with R7050, an inhibitor of TNFR1 signalling (Cheng et al., 2021), attenuated the TNFα‐mediated rise in [Ca 2+ ] i in DRG neurons isolated from wild‐type (WT) mice ( P = 0.035, one‐way ANOVA with Holm–Šídák's multiple comparisons test; n = 5, N = 5; Fig. 2) and reduced the proportion of TNFα‐sensitive neurons ( P = 0.004, one‐way ANOVA with Holm–Šídák's multiple comparisons test; n = 5, N = 5; Fig.…”

Erythropoietin in Optic Neuropathies: Current Future Strategies for Optic Nerve Protection and Repair

“…Ca 2+ acts as second messenger to activate downstream signaling pathways leading to RGCs apoptosis [73]. TNF-α and TNF-α receptor 1 signaling could also induce RGC hyper-excitability by upregulating Na + channels, which contribute to RGCs apoptosis in glaucoma [74]. In our previous study, we cultured RGCs from adult rats in a medium containing neurotrophic factors [12].…”